Crystalline forms of a JAK inhibitor compound

ABSTRACT

The present invention provides crystalline hydrates of the oxalate and succinate salts of 5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol. The invention also provides pharmaceutical compositions comprising such crystalline hydrates, methods of using such crystalline hydrates to treat respiratory and other diseases, and processes useful for preparing such crystalline oxalate and succinate hydrates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No62/492,571, filed on May 1, 2017, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to crystalline salt forms of a JAK inhibitorcompound useful for treating respiratory and other diseases. Theinvention is also directed to pharmaceutical compositions comprisingsuch compound, methods of using the salt forms to treat, for example,respiratory and ocular diseases, and processes and intermediates usefulfor preparing such crystalline salt forms.

State of the Art

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of a large number of disease orconditions, particularly those diseases characterized by inflammation.Many of the cytokines implicated in disease act through signalingpathways dependent upon the Janus family of tyrosine kinases (JAKs),which signal through the Signal Transducer and Activator ofTranscription (STAT) family of transcription factors.

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated withinflammation.

Asthma is a chronic disease of the airways for which there are nopreventions or cures. The disease is characterized by inflammation,fibrosis, hyperresponsiveness, and remodeling of the airways, all ofwhich contribute to airflow limitation. An estimated 300 million peopleworldwide suffer from asthma and it is estimated that the number ofpeople with asthma will grow by more than 100 million by 2025. Althoughmost patients can achieve control of asthma symptoms with the use ofinhaled corticosteroids that may be combined with a leukotriene modifierand/or a long acting beta agonist, there remains a subset of patientswith severe asthma whose disease is not controlled by conventionaltherapies. Cytokines implicated in asthma inflammation which signalthrough the JAK-STAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9,IL-11, IL-13, IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP),interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor(GM-CSF). Inflammation of the airways is characteristic of otherrespiratory diseases in addition to asthma. Chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, and bronchiolitis obliterans are also respiratory tractdiseases in which the pathophysiology is believed to be related toJAK-signaling cytokines.

Inflammation plays a prominent role in many ocular diseases, includinguveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, and atopic keratoconjunctivitis.Uveitis encompasses multiple intraocular inflammatory conditions and isoften autoimmune, arising without a known infectious trigger. Thecondition is estimated to affect about 2 million patients in the US. Insome patients, the chronic inflammation associated with uveitis leads totissue destruction, and it is the fifth leading cause of blindness inthe US. Cytokines elevated in uveitis patients' eyes that signal throughthe JAK-STAT pathway include IL-2, IL-4, IL-5, IL-6, IL-10, IL-23, andIFN-γ. (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744;Ooi et al, Clinical Medicine and Research, 2006, 4, 294-309). Existingtherapies for uveitis are often suboptimal, and many patients are poorlycontrolled. Steroids, while often effective, are associated withcataracts and increased intraocular pressure/glaucoma.

Diabetic retinopathy (DR) is caused by damage to the blood vessels inthe retina. It is the most common cause of vision loss among people withdiabetes. Angiogenic as well as inflammatory pathways play an importantrole in the disease. Often, DR will progress to diabetic macular edema(DME), the most frequent cause of visual loss in patients with diabetes.The condition is estimated to affect about 1.5 million patients in theUS alone, of whom about 20% have disease affecting both eyes. Cytokineswhich signal through the JAK-STAT pathway, such as IL-6, as well asother cytokines, such as IP-10 and MCP-1 (alternatively termed CCL2),whose production is driven in part by JAK-STAT pathway signaling, arebelieved to play a role in the inflammation associated with DR/DME(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al.,American Journal of Opthalmology, 2011, 152, 686-694; Owen and Hartnett,Curr Diab Rep, 2013, 13, 476-480; Cheung et al, Molecular Vision, 2012,18, 830-837; Dong et al, Molecular Vision, 2013, 19, 1734-1746; Funatsuet al, Ophthalmology, 2009, 116, 73-79). The existing therapies for DMEare suboptimal: intravitreal anti-VEGF treatments are only effective ina fraction of patients and steroids are associated with cataracts andincreased intraocular pressure.

Dry eye disease (DED) is a multifactorial disorder that affectsapproximately 5 million patients in the US. Ocular surface inflammationis believed to play an important role in the development and propagationof this disease. Elevated levels of cytokines such as IL-1, IL-2, IL-4,IL-5, IL-6, and IFN-γ have been noted in the ocular fluids of patientswith DED. (Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and thelevels often correlated with disease severity. Age-related maculardegeneration and atopic keratoconjunctivitis are also thought to beassociated with JAK-dependent cytokines.

Commonly assigned U.S. application Ser. No. 15/341,226, filed Nov. 2,2016 discloses diamino compounds useful as JAK inhibitors. Inparticular, the compound5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1)

is specifically disclosed in the application as a potent pan-JAKinhibitor.

To effectively use this compound as a therapeutic agent, it would bedesirable to have a crystalline solid-state salt form. For example, itwould be highly desirable to have a physical form that is thermallystable at reasonably high temperature, thereby facilitating processingand storage of the material. Crystalline solids are generally preferredover amorphous forms, for enhancing purity and stability of themanufactured product. However, the formation of crystalline forms oforganic compounds is highly unpredictable. No reliable methods exist forpredicting which, if any, form of an organic compound will becrystalline. Moreover, no methods exist for predicting which, if any,crystalline form will have the physically properties desired for use aspharmaceutical agents.

No crystalline salt forms of compound 1 have previously been reported.Accordingly, a need exists for crystalline salt forms of compound 1.

SUMMARY OF THE INVENTION

The present invention provides crystalline hydrates of the oxalate andsuccinate salts of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1).

The crystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolhas been found to have a melting temperature in the range of about 266°C. to about 276° C. and to exhibit total moisture uptake of about 1%when exposed to a range of relative humidity between about 30% and about90% at room temperature.

The crystalline hydrate of the succinate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolhas been found to have a melting temperature in the range of about 180°C. to about 190° C. and to exhibit total moisture uptake of about 2%when exposed to a range of relative humidity between about 5% and about90% at room temperature.

Among other uses, the crystalline solid forms of the invention areexpected to be useful for preparing pharmaceutical compositions fortreating or ameliorating disease amenable to treatment with a JAKinhibitor, in particular respiratory disease. Accordingly, in another ofits composition aspects, the invention provides a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier and anactive agent selected from the crystalline hydrate of the oxalate saltof5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenoland the crystalline hydrate of the succinate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.

The invention also provides a method of treating respiratory disease, inparticular, asthma, in a mammal, the method comprising administering tothe mammal a crystalline solid form or a pharmaceutical composition ofthe invention. In separate and distinct aspects, the invention alsoprovides synthetic processes useful for preparing the crystalline formsof the invention.

The invention further provides a method of treating an ocularinflammatory disease in a mammal, the method comprising administering tothe eye of the mammal, a crystalline solid form or a pharmaceuticalcomposition of the invention.

The invention also provides a crystalline solid form of the invention asdescribed herein for use in medical therapy, as well as the use of acrystalline solid form of the invention in the manufacture of aformulation or medicament for treating respiratory disease in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of thecrystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(hereinafter ‘oxalate hydrate’).

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram of theoxalate hydrate of the invention.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of the oxalatehydrate of the invention.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of the oxalatehydrate of the invention observed at a temperature of about 25° C.

FIG. 5 shows a powder x-ray diffraction (PXRD) pattern of thecrystalline succinate hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(hereinafter ‘succinate hydrate’).

FIG. 6 shows a differential scanning calorimetry (DSC) thermogram of thesuccinate hydrate of the invention.

FIG. 7 shows a thermal gravimetric analysis (TGA) plot of the succinatehydrate of the invention.

FIG. 8 shows a dynamic moisture sorption (DMS) isotherm of the succinatehydrate of the invention observed at a temperature of about 25° C.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” means preventing, ameliorating orsuppressing the medical condition, disease or disorder being treated(e.g., a respiratory disease) in a patient (particularly a human); oralleviating the symptoms of the medical condition, disease or disorder.

The term “hydrate” means a complex or aggregate, typically incrystalline form, formed by molecules of water and the compound of theinvention where the ratio of water molecules to compound molecules maybe less than 1:1 or more than 1:1.

The term “about” means±5 percent of the specified value.

It must be noted that, as used in the specification and appended claims,the singular forms “a”, “an”, “one”, and “the” may include pluralreferences, unless the content clearly dictates otherwise.

Naming Convention

Compound 1 is designated as5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolaccording to IUPAC conventions as implemented in ChemDraw software(PerkinElmer, Inc., Cambridge, Mass.).

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of compound 1 exists in tautomeric forms, illustratedbelow for a fragment of the compound of Example 1

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the imidazole portion:2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(structure A) vs.2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine(structure B). It will be understood that although structures are shown,or named, in a particular form, the invention also includes the tautomerthereof.

Crystalline Forms of the Invention

In one aspect, the invention provides the crystalline hydrate of theoxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1).

In one aspect, the crystalline oxalate hydrate is characterized by apowder X-ray diffraction (PXRD) pattern having significant diffractionpeaks, among other peaks, at 2θ values of 6.77±0.20, 12.13±0.20,13.54±0.20, 17.23±0.20, and 18.00±0.20. The crystalline oxalate hydratemay be further characterized by a PXRD pattern having two or moreadditional diffraction peaks, including three or more additionaldiffraction peaks at 2θ values selected from 11.56±0.20, 14.29±0.20,19.51±0.20, 21.38±0.20, and 23.63±0.20. In another aspect, thecrystalline oxalate hydrate is characterized by a PXRD pattern havingdiffraction peaks at 20 values of 6.77±0.20, 11.56±0.20, 12.13±0.20,13.54±0.20, 14.29±0.20, 17.23±0.20, 18.00±0.20, 19.51±0.20, 21.38±0.20,and 23.63±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineoxalate hydrate is characterized by a powder x-ray diffraction patternin which the peak positions are substantially in accordance with thoseshown in FIG. 1.

In another aspect, the crystalline oxalate hydrate is characterized byits behavior when exposed to high temperature. As demonstrated in FIG.2, the differential scanning calorimetry (DSC) trace recorded at aheating rate of 10° C. per minute exhibits a desolvation endotherm withan onset at about 59° C. and a peak at about 97° C. and a peak inendothermic heat flow, identified as a melt transition, in the range ofabout 266° C. to about 276° C. including between about 268° C. and about273° C. The thermal gravimetric analysis (TGA) trace of FIG. 3 shows adesolvation onset at a temperature of about 26° C. and a decompositiononset at a temperature of about 250° C. Taken together, the DSC and TGAtraces suggest the melt transition is accompanied by decomposition. TheTGA profile shows a weight loss of about 5.5% between about 25° C. andabout 75° C.

The present crystalline oxalate hydrate has been demonstrated to have areversible sorption/desorption profile with a slight propensity forhygroscopicity. The oxalate hydrate exhibited total moisture uptake ofabout 1% when exposed to a range of relative humidity between about 30%and about 90% at room temperature as shown in FIG. 4. A reversiblehydration/dehydration transition was observed between about 0% and 15%relative humidity. No hysteresis was observed in two cycles of sorptionand desorption.

In another aspect, the invention provides the crystalline hydrate of thesuccinate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1)

In one aspect, the crystalline succinate hydrate is characterized by apowder X-ray diffraction (PXRD) pattern having significant diffractionpeaks, among other peaks, at 2θ values of 4.81±0.20, 9.66±0.20,14.93±0.20, and 16.78±0.20. The crystalline succinate hydrate may befurther characterized by a PXRD pattern having two or more additionaldiffraction peaks, including three or more additional diffraction peaksat 2θ values selected from 10.46±0.20, 16.21±0.20, 17.45±0.20,22.87±0.20, and 24.77±0.20. In another aspect, the crystalline oxalatehydrate is characterized by a PXRD pattern having diffraction peaks at20 values of 4.81±0.20, 9.66±0.20, 10.46±0.20, 14.93±0.20, 16.21±0.20,16.78±0.20, 17.45±0.20, 22.87±0.20, and 24.77±0.20. In yet anotheraspect, the crystalline succinate hydrate is characterized by a powderx-ray diffraction pattern in which the peak positions are substantiallyin accordance with those shown in FIG. 5.

The crystalline succinate hydrate is also characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 6, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits two desolvation endotherms: one with anonset at about 20° C. and a peak at about 50° C. and a seconddesolvation endotherm with an onset at about 103° C. and a peak at about129° C. The DSC trace further exhibits a peak in endothermic heat flow,identified as a melt transition, in the range of about 180° C. to about190° C. including between about 183° C. and about 188° C. The thermalgravimetric analysis (TGA) trace of FIG. 7 shows a decomposition onsetat a temperature of about 200° C.

The crystalline succinate hydrate has been demonstrated to have areversible sorption/desorption profile with a slight propensity forhygroscopicity. The succinate hydrate exhibited total moisture uptake ofabout 2% when exposed to a range of relative humidity between about 5%and about 90% at room temperature as shown in FIG. 8. No hysteresis wasobserved in two cycles of sorption and desorption.

Synthetic Procedures

Compound 1, can be prepared from readily available starting materialsusing the procedures described in the Examples below, or using theprocedures described in the commonly-assigned U.S. application listed inthe Background section of this application.

The crystalline oxalate hydrate of the invention is convenientlyprepared by dissolving an equimolar mixture of compound 1 and oxalicacid in a 1:1 mixture of tetrahydrofuran and water at room temperaturefollowed by the addition of a 1:1:2 mixture oftetrahydrofuran:water:acetonitrile, as an antisolvent to produce asuspension. The resulting reaction mixture is stirred for about one dayat room temperature, washed with acetonitrile, and dried to provide thecrystalline hydrate form.

The present crystalline succinate hydrate may be prepared by a threestage process. First, an equimolar mixture of compound 1 and succinicacid is suspended in isopropanol and stirred for about one day at roomtemperature. The resulting solids are filtered, washed with isopropanol,and dried to provide a first intermediate crystalline solid. Second, theisolated first intermediate crystalline solid is dried at about 150° C.for about 30 minutes to provide a second intermediate crystalline solid.Third, the second intermediate solid is equilibrated under about 80% to90% relative humidity for about one day at room temperature to providethe crystalline succinate hydrate form.

Accordingly in a method aspect, the invention provides a method ofpreparing the crystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,the method comprising (a) dissolving a 1:1 mixture of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol:oxalic acid in a 1:1 mixture of tetrahydrofuran:water at roomtemperature, (b) adding a 1:1:2 mixture oftetrahydrofuran:water:acetonitrile to produce a suspension, (c) stirringthe suspension for about one day, and (d) isolating the crystallineoxalate hydrate from the suspension.

Pharmaceutical Compositions

The crystalline solid forms of the invention are typically used in theform of a pharmaceutical composition or formulation. Such pharmaceuticalcompositions may advantageously be administered to a patient byinhalation. In addition, pharmaceutical compositions may be administeredby any acceptable route of administration including, but not limited to,oral, topical (including transdermal), rectal, nasal, and parenteralmodes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a crystallineoxalate hydrate or crystalline succinate hydrate of compound 1.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired. When discussingcompositions and uses thereof, the crystalline solid forms of theinvention may also be referred to herein as the “active agent”.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the crystalline forms of the presentinvention. Those skilled in the art will recognize, however, that apharmaceutical composition may contain more than a therapeuticallyeffective amount, i.e., bulk compositions, or less than atherapeutically effective amount, i.e., individual unit doses designedfor multiple administration to achieve a therapeutically effectiveamount.

Typically, such pharmaceutical compositions will contain from about 0.01to about 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Maryland (2000); and H. C. Ansel et al., PharmaceuticalDosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams& White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

In one aspect, the pharmaceutical composition is suitable for inhaledadministration. Pharmaceutical compositions for inhaled administrationare typically in the form of an aerosol or a powder. Such compositionsare generally administered using inhaler delivery devices, such as a drypowder inhaler (DPI), a metered-dose inhaler (MDI), a nebulizer inhaler,or a similar delivery device.

In a particular embodiment, the pharmaceutical composition isadministered by inhalation using a dry powder inhaler. Such dry powderinhalers typically administer the pharmaceutical composition as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free-flowing powder composition, thetherapeutic agent is typically formulated with a suitable excipient suchas lactose, starch, mannitol, dextrose, polylactic acid (PLA),polylactide-co-glycolide (PLGA) or combinations thereof. Typically, thetherapeutic agent is micronized and combined with a suitable carrier toform a composition suitable for inhalation.

A representative pharmaceutical composition for use in a dry powderinhaler comprises lactose and a crystalline solid form of the inventionin micronized form. Such a dry powder composition can be made, forexample, by combining dry milled lactose with the therapeutic agent andthen dry blending the components. The composition is then typicallyloaded into a dry powder dispenser, or into inhalation cartridges orcapsules for use with a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are described in the art and examplesof such devices are commercially available. For example, representativedry powder inhaler delivery devices or products include Aeolizer(Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler(GlaxoSmithKline); Diskus/Accuhaler (GlaxoSmithKline); Ellipta(GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps(Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi);Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler(Aventis); and the like.

In another particular embodiment, the pharmaceutical composition isadministered by inhalation using a metered-dose inhaler. Suchmetered-dose inhalers typically discharge a measured amount of atherapeutic agent using a compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the therapeutic agent ina liquefied propellant. Any suitable liquefied propellant may beemployed including hydrofluoroalkanes (HFAs), such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and chlorofluorocarbons,such as CCl₃F. In a particular embodiment, the propellant ishydrofluoroalkanes. In some embodiments, the hydrofluoroalkaneformulation contains a co-solvent, such as ethanol or pentane, and/or asurfactant, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof the invention; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant. Such compositions are typically prepared by addingchilled or pressurized hydrofluoroalkane to a suitable containercontaining the therapeutic agent, ethanol (if present) and thesurfactant (if present). To prepare a suspension, the therapeutic agentis micronized and then combined with the propellant. The composition isthen loaded into an aerosol canister, which typically forms a portion ofa metered-dose inhaler device.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are described in the art and examples of suchdevices are commercially available. For example, representativemetered-dose inhaler devices or products include AeroBid Inhaler System(Forest Pharmaceuticals); Atrovent Inhalation Aerosol (BoehringerIngelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); ProventilInhaler (Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); andthe like.

In another particular aspect, the pharmaceutical composition isadministered by inhalation using a nebulizer inhaler. Such nebulizerdevices typically produce a stream of high velocity air that causes thepharmaceutical composition to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the therapeutic agent can be dissolved in a suitablecarrier to form a solution. Alternatively, the therapeutic agent can bemicronized or nanomilled and combined with a suitable carrier to form asuspension.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises a solution or suspension comprising from about 0.05μg/mL to about 20 mg/mL of a compound of the invention and excipientscompatible with nebulized formulations. In one embodiment, the solutionhas a pH of about 3 to about 8.

Nebulizer devices suitable for administering therapeutic agents byinhalation are described in the art and examples of such devices arecommercially available. For example, representative nebulizer devices orproducts include the Respimat Softmist Inhaler (Boehringer Ingelheim);the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI LC PlusReusable Nebulizer (Pari GmbH); and the like.

In yet another aspect, the pharmaceutical compositions of the inventionmay alternatively be prepared in a dosage form intended for oraladministration. Suitable pharmaceutical compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; or as a solution or a suspension inan aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilliquid emulsion; or as an elixir or syrup; and the like; each containinga predetermined amount of a compound of the present invention as anactive ingredient.

When intended for oral administration in a solid dosage form, thepharmaceutical compositions of the invention will typically comprise theactive agent and one or more pharmaceutically-acceptable carriers, suchas sodium citrate or dicalcium phosphate. Optionally or alternatively,such solid dosage forms may also comprise: fillers or extenders,binders, humectants, solution retarding agents, absorption accelerators,wetting agents, absorbents, lubricants, coloring agents, and bufferingagents. Release agents, wetting agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the pharmaceutical compositions of the invention.

The crystalline solid forms may also be formulated as a sterile aqueoussuspension or solution for ocular injection. Useful excipients that maybe included in such an aqueous formulation include polysorbate 80,carboxymethylcellulose, potassium chloride, calcium chloride, magnesiumchloride, sodium acetate, sodium citrate, histidine, α-α-trehalosedihydrate, sucrose, polysorbate 20, hydroxypropyl-β-cyclodextrin, andsodium phosphate. Benzyl alcohol may serve as a preservative and sodiumchloride may be included to adjust tonicity. In addition, hydrochloricacid and/or sodium hydroxide may be added to the solution for pHadjustment. Aqueous formulations for ocular injection may be prepared aspreservative-free.

Alternative formulations may also include controlled releaseformulations, liquid dosage forms for oral administration, transdermalpatches, and parenteral formulations. Conventional excipients andmethods of preparation of such alternative formulations are described,for example, in the reference by Remington, supra.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Dry Powder Composition

A micronized solid form of the invention (1 g) is blended with milledlactose (25 g). This blended mixture is then loaded into individualblisters of a peelable blister pack in an amount sufficient to providebetween about 0.1 mg to about 4 mg of the compound of formula I perdose. The contents of the blisters are administered using a dry powderinhaler.

Dry Powder Composition

A micronized solid form of the invention (1 g) is blended with milledlactose (20 g) to form a bulk composition having a weight ratio ofcompound to milled lactose of 1:20. The blended composition is packedinto a dry powder inhalation device capable of delivering between about0.1 mg to about 4 mg of the compound of formula I per dose.

Metered-Dose Inhaler Composition

A micronized solid form of the invention (10 g) is dispersed in asolution prepared by dissolving lecithin (0.2 g) in demineralized water(200 mL). The resulting suspension is spray dried and then micronized toform a micronized composition comprising particles having a meandiameter less than about 1.5 μ. The micronized composition is thenloaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1mg to about 4 mg of the compound of formula I per dose when administeredby the metered dose inhaler.

Nebulizer Composition

A solid form of the invention (25 mg) is dissolved in a solutioncontaining 1.5-2.5 equivalents of hydrochloric acid, followed byaddition of sodium hydroxide to adjust the pH to 3.5 to 5.5 and 3% byweight of glycerol. The solution is stirred well until all thecomponents are dissolved. The solution is administered using a nebulizerdevice that provides about 0.1 mg to about 4 mg of the compound offormula I per dose.

Aqueous Formulation for Ocular Injection

Each mL of a sterile aqueous suspension includes from 5 mg to 50 mg of asolid form of the invention, sodium chloride for tonicity, 0.99% (w/v)benzyl alcohol as a preservative, 0.75% carboxymethylcellulose sodium,and 0.04% polysorbate. Sodium hydroxide or hydrochloric acid may beincluded to adjust pH to 5 to 7.5.

Aqueous Formulation for Ocular Injection

A sterile preservative-free aqueous suspension includes from 5 mg/mL to50 mg/mL of a solid form of the invention in 10 mM sodium phosphate, 40mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose.

Utility

The present compound,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-y0-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,(compound 1), has been shown to be a potent inhibitor of the JAK familyof enzymes: JAK1, JAK2, JAK3, and TYK2.

Respiratory Diseases

In addition, as described in the assays below, compound 1 hasdemonstrated potent inhibition of pro-inflammatory and pro-fibroticcytokines implicated in asthma and other respiratory diseases. Theabsorption and distribution of the compound has been profiled inpreclinical assays. In mouse the compound exhibited exposure in lungabout 55 times greater than the exposure in plasma. Importantly, theconcentration of compound 1 in the mouse lung has been shown tocorrelate with a predicted pharmacodynamic effect of JAK enzymeinhibition. In particular, the compounds has been shown to inhibit aneffect of the pro-inflammatory cytokine IL-13 in mouse lung tissue.Specifically, the compound demonstrated inhibition of IL-13-inducedphosphorylation of STAT6 in lung tissue which provides evidence of locallung JAK target engagement in vivo. This effect has been observed whenthe pro-inflammatory cytokine IL-13 is administered 4 hours afteradministration of the test compound, providing further evidence ofsignificant retention in the lung.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829,-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161.) Accordingly, the compounds of the invention areexpected to be useful for the treatment of inflammatory respiratorydisorders, in particular, asthma. Inflammation and fibrosis of the lungis characteristic of other respiratory diseases in addition to asthmasuch as chronic obstructive pulmonary disease (COPD), cystic fibrosis(CF), pneumonitis, interstitial lung diseases (including idiopathicpulmonary fibrosis), acute lung injury, acute respiratory distresssyndrome, bronchitis, emphysema, and bronchiolitis obliterans. Thepresent compounds, therefore, are also expected to be useful for thetreatment of chronic obstructive pulmonary disease, cystic fibrosis,pneumonitis, interstitial lung diseases (including idiopathic pulmonaryfibrosis), acute lung injury, acute respiratory distress syndrome,bronchitis, emphysema and bronchiolitis obliterans. As described above,for treatment of respiratory diseases, solid forms are particular usefulfor administration by inhalation.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal a therapeutically-effective amount of acompound of the invention or of a pharmaceutical composition comprisinga pharmaceutically-acceptable carrier and a solid form of the invention.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema or bronchiolitis obliterans. In another aspect, therespiratory disease is asthma or chronic obstructive pulmonary disease.In another aspect, the solid forms of the invention are administered byinhalation.

The invention further provides a method of treating asthma in a mammal,the method comprising administering to the mammal a solid form of theinvention or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a solid form of the invention.

When used to treat asthma, the compounds of the invention will typicallybe administered in a single daily dose or in multiple doses per day,although other forms of administration may be used. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

In addition to having demonstrated potent inhibition of cytokinesassociated with inflammation, compound 1 has demonstrated inhibition ofT cell activation and activity in rodent lung eosinophilia andneutrophilia assays. Therefore, the solid forms of the invention arebelieved to useful for the treatment of additional respiratoryconditions.

The additional respiratory conditions include lung infections,helminthic infections, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, and infiltrative pulmonarydisease. The solid forms are also believed to be useful for thetreatment of drug-induced pneumonitis, fungal induced pneumonitis,allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis,eosinophilic granulomatosis with polyangiitis, idiopathic acuteeosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,hypereosinophilic syndrome, Loffler syndrome, bronchiolitis obliteransorganizing pneumonia, and immune-checkpoint-inhibitor inducedpneumonitis.

JAK-signaling cytokines also play a major role in the activation of Tcells, a sub-type of immune cells that is central to many immuneprocesses. Pathological T cell activation is critical in the etiology ofmultiple respiratory diseases. Autoreactive T cells play a role inbronchiolitis obliterans organizing pneumonia (also termed COS). Similarto COS the etiology of lung transplant rejections is linked to anaberrant T cell activation of the recipients T cells by the transplanteddonor lung. Lung transplant rejections may occur early as Primary GraftDysfunction (PGD), organizing pneumonia (OP), acute rejection (AR) orlymphocytic bronchiolitis (LB) or they may occur years after lungtransplantation as Chronic Lung Allograft Dysfunction (CLAD). CLAD waspreviously known as bronchiolitis obliterans (BO) but now is considereda syndrome that can have different pathological manifestations includingBO, restrictive CLAD (rCLAD or RAS) and neutrophilic allograftdysfunction. Chronic lung allograft dysfunction (CLAD) is a majorchallenge in long-term management of lung transplant recipients as itcauses a transplanted lung to progressively lose functionality (Gauthieret al., Curr Transplant Rep., 2016, 3(3), 185-191). CLAD is poorlyresponsive to treatment and therefore, there remains a need foreffective compounds capable of preventing or treating this condition.

Several JAK-dependent cytokines such as IFNγ and IL-5 are up-regulatedin CLAD and lung transplant rejection (Berastegui et al, ClinTransplant. 2017, 31, e12898). Moreover, high lung levels of CXCR3chemokines such as CXCL9 and CXCL10 which are downstream ofJAK-dependent IFN signaling, are linked to worse outcomes in lungtransplant patients (Shino et al, PLOS One, 2017, 12 (7), e0180281).Systemic JAK inhibition has been shown to be effective in kidneytransplant rejection (Vicenti et al., American Journal ofTransplantation, 2012, 12, 2446-56). Therefore, JAK inhibitors have thepotential to be effective in treating or preventing lung transplantrejection and CLAD. Similar T cell activation events as described as thebasis for lung transplant rejection also are considered the main driverof lung graft-versus-host disease (GVHD) which can occur posthematopoietic stem cell transplants. Similar to CLAD, lung GVHD is achronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). As systemic JAK inhibition is associated withserious adverse events and a small therapeutic index, the need remainsfor an inhaled lung-directed, non-systemic JAK inhibitor to preventand/or treat lung transplant rejection or lung GVHD.

Accordingly, the invention further provides a method of treating theadditional respiratory conditions described above in a mammal, themethod comprising administering to the mammal a solid form of theinvention or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a solid form of the invention.

Ocular Diseases

Many ocular diseases have been shown to be associated with elevations ofproinflammatory cytokines that rely on the JAK-STAT pathway. Since thecompound of the invention exhibits potent inhibition at all four JAKenzymes, it is expected to potently inhibit the signaling and pathogeniceffects of numerous cytokines (such as IL-6, IL-2 and IFN-γ), thatsignal through JAK, as well as to prevent the increase in othercytokines (such as MCP-1 and IP-10), whose production is driven byJAK-STAT pathway signaling.

In particular, Compound 1 exhibited pIC₅₀ values of 6.7 or greater (IC₅₀values of 200 nM or less) for inhibition of IL-2, IL-4, IL-6, and IFNγsignaling in the cellular assays described in Assays 3 to 7, includingassays registering inhibition of the downstream effects of cytokineelevation.

The pharmacokinetic study of Assay 12 demonstrated sustained exposure inrabbit eyes after a single intravitreal injection of a suspension of thecrystalline compound 1 of example 2, and a concentration in plasma atleast three orders of magnitude lower than that observed in vitreoustissue. Assays 13 and 14 demonstrated a pharmacodynamic effect of thecompound in rats and rabbits.

The solid forms of the invention, therefore, are expected to bebeneficial in a number of ocular diseases that include, but are notlimited to, uveitis, diabetic retinopathy, diabetic macular edema, dryeye disease, age-related macular degeneration, and atopickeratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res,2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol,2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., AmericanJournal of Opthalmology, 2011, 152, 686-694), dry eye disease (Stevensonet al, Arch Ophthalmol, 2012, 130, 90-100), and age-related maculardegeneration (Knickelbein et al, Int Ophthalmol Clin, 2015, 55(3),63-78) are characterized by elevation of certain pro-inflammatorycytokines that signal via the JAK-STAT pathway. Accordingly, the solidforms of the invention may be able to alleviate the associated ocularinflammation and reverse disease progression or provide symptom relief.

Retinal vein occlusion (RVO) is a highly prevalent visually disablingdisease. Obstruction of retinal blood flow can lead to damage of theretinal vasculature, hemorrhage, and tissue ischemia. Although thecauses for RVO are multifactorial, both vascular as well as inflammatorymediators have been shown to be important (Deobhakta et al,International Journal of Inflammation, 2013, article ID 438412).Cytokines which signal through the JAK-STAT pathway, such as IL-6 andIL-13, as well as other cytokines, such as MCP-1, whose production isdriven in part by JAK-STAT pathway signaling, have been detected atelevated levels in ocular tissues of patients with RVO (Shchuko et al,Indian Journal of Ophthalmology, 2015, 63(12), 905-911). Accordingly,the solid forms of the invention may be able to alleviate the associatedocular inflammation and reverse disease progression or provide symptomrelief in this disease. While many patients with RVO are treated byphotocoagulation, this is an inherently destructive therapy. Anti-VEGFagents are also used, but they are only effective in a fraction ofpatients. Steroid medications that reduce the level of inflammation inthe eye (Triamcinolone acetonide and dexamethasone implants) have alsobeen shown to provide beneficial results for patients with certain formsof RVO, but they have also been shown to cause cataracts and increasedintraocular pressure/glaucoma.

In one aspect, therefore, the invention provides a method of treating anocular disease in a mammal, the method comprising administering a solidform of the invention to the eye of the mammal. In one aspect, theocular disease is uveitis, diabetic retinopathy, diabetic macular edema,dry eye disease, age-related macular degeneration, or atopickeratoconjunctivitis. In one aspect, the ocular disease is retinal veinocclusion.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

ACN=acetonitrile

CPME=cyclopentyl methyl ether

DCM=dichloromethane

DIPEA=N,N-diisopropylethylamine

DMAc=dimethylacetamide

DMF=N,N-dimethylformamide

EtOAc=ethyl acetate

h=hour(s)

IPAc=isopropylacetate

KOAc=potassium acetate

MeOH=methanol

MeTHF=2-methyltetrahydrofuran

min=minute(s)

MTBE=methyl tent-butyl ether

NMP=N-methyl-2-pyrrolidone

Pd(amphos)₂Cl₂=bis(di-tert-butyl(4-dimethylaminophenyl)-phosphine)dichloropalladium(II)

Pd(dppf)Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)dipalladium(II)

Pd(PPh₃)₄=tetrakis(triphenylphosphine)palladium(0)

Pd(t-Bu₃P)₂=bis(tri-tert-butylphosphine) palladium(0)

RT=room temperature

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2]bi[[1,3,2]dioxaborolanyl]

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions

-   Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm 21×250 or C14, 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA B=ACN+0.05% TFA,-   Injection volume: (100-1500 μL)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL . A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytic HPLC Conditions

Method A

-   Column: Agilent Zorbax Bonus-RP C18, 150×4.60 nm, 3.5 micron-   Column temperature: 40° C.-   Flow rate: 1.5 mL/min-   Injection volume: 5 μL-   Sample preparation: Dissolve in 1:1 ACN:1 M HCl-   Mobile Phases: A=Water: TFA (99.95:0.05) B=ACN:TFA (99.95:0.05)-   Detector wavelength: 254 nm and 214 nm-   Gradient: 26 min total (time (min)/ % B): 0/5, 18/90, 22/90,    22.5/90, 26/5    Method B-   Column: Agilent Poroshell 120 Bonus-RP, 4.6×150 mm, 2.7 μm-   Column temperature: 30° C.-   Flow rate: 1.5 mL/min-   Injection volume: 10 μL-   Mobile Phases: A=ACN:Water:TFA (2:98:0.1) B=ACN:Water:TFA    (90:10:0.1)-   Sample preparation: Dissolve in Mobile phase B-   Detector wavelength: 254 nm and 214 nm-   Gradient: 60 min total (time (min)/ % B): 0/0, 50/100, 55/100,    55.1/0, 60/0    Method C-   Column: Agilent Poroshell 120 Bonus-RP, 4.6×150 mm, 2.7 μm-   Column temperature: 30° C.-   Flow rate: 1.5 mL/min-   Injection volume: 10 μL-   Mobile Phases: A=ACN:Water:TFA (2:98:0.1) B=ACN:Water:TFA    (90:10:0.1)-   Sample preparation: Dissolve in Mobile phase B (0.15 mL) then dilute    with Mobile phase A (0.85 mL)-   Detector wavelength: 245 nm-   Gradient: 46 min total (time (min)/ % B): 0/0, 25/50, 35/100,40/100,    40.1/0, 46/0

Preparation 1: 1-benzyl-4-imino-1,4-dihydropyridin-3-amine

A mixture of pyridine-3,4-diamine (445 g, 4.078 mol) and ACN (11.0 L)was stirred for 80 min from 25° C. to 15° C. Benzyl bromide (485 mL,4.078 mol) was added over 20 min and the reaction mixture was stirred at20° C. overnight. The reaction mixture was cooled to 10° C. andfiltered. To the reactor was added ACN (3 L), which was cooled to 10° C.The cake was washed with the reactor rinse and washed again with ACN (3L) warmed to 25° C. The solid was dried on the filter for 24 h undernitrogen, at 55° C. under vacuum for 2 h and then at RT overnight andfor 4 d to provide the HBr salt of the title compound (1102.2 g, 3.934mol, 96% yield). HPLC Method A Retention time 4.12 min.

Preparation 2:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

A solution of 6-bromo-1H-indazole-3-carbaldehyde (550 g, 2.444 mol),1-benzyl-4-imino-1,4-dihydropyridin-3-amine HBr (721 g, 2.333 mol) andDMAc (2.65 L) was stirred for 60 min and sodium bisulfite (257 g, 2.468mol) was added. The reaction mixture was heated to 135° C. and held for3 h, and allowed to cool to 20° C. and held at 20° C. overnight.Acetonitrile (8 L) was added and the reaction mixture was stirred for 4h at 15° C. The slurry was filtered on a pressure filter at mediumfiltration rate. To the reactor was added ACN (1 L) The cake was washedwith the ACN reactor wash and dried under nitrogen overnight and thenunder vacuum at 50° C. for 24 h to provide the HBr salt of the titlecompound (1264 g, 2.444 mol, 100% yield, 94% purity) as a dense wetbeige/brown solid. HPLC Method A Retention time 8.77 min.

A mixture of the product of the previous step (1264 g, 2.444 mol), MeTHF(6 L) and water (2.75 L) was heated to 65° C. and sodium hydroxide 50 wt% (254 g, 3.177 mol) was added over 5 min and the reaction mixture wasstirred at 65° C. for 1 h, cooled to RT, then to 5° C. and held for 2 h.The slurry was filtered and the reactor and cake were washed with MeTHF(1 L). The resulting beige to yellow solid was dried on the filter undernitrogen for 3 d to provide the title compound (475 g, 1.175 mmol, 48%yield) as a beige/yellow solid. The mother liquor (about 8 L) wasconcentrated to about 2 L, whereupon solids began to crash out., Theslurry was heated to 50° C., held for 2 h, cooled to 5° C. over 2 h,stirred overnight, and filtered. The cake was washed with MeTHF (100 mL)and dried overnight under vacuum at 40° C. to provide additional titlecompound (140 g, 0.346 mol, 14% yield).

A mixture of the total product of the previous step, combined with theproduct of a second batch at the same scale (1500 g, 3.710 mol) andMeTHF (4 L) was stirred at 20° C. for 2 h and filtered. The reactor andcake were washed with MeTHF (1.5 L). The resulting beige to yellow solidwas dried under nitrogen for 3 d to provide the title compound as abeige yellow solid (1325 g, 3.184 mol, 86% yield (overall 68% yield),97% purity). HPLC Method A Retention time 8.77 min

Preparation 3:5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a 15 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (440 g,1.088 mol) followed by MeTHF (4.5 L), methanol (2.25 L) and water (1.125L). The slurry was cooled to 20° C., stirred for 1 h, and NaBH₄ (247 g,6.530 mol) was added. The reaction mixture was stirred at 25° C. for 18h. Water (1.125 L) was added followed by 20 wt %. sodium chloridesolution (1.125 L) and the mixture was stirred for 30 min and the layersallowed to separate. The aqueous layer was drained. A premixed solutionof NaOH (522 g) and water (5 L) was added and the reaction mixture wasstirred for 60 min; the layers were allowed to separate and the aqueouslayer was drained. Two additional batches at the same scale wereprepared.

The organic layer from one batch was concentrated under reduced pressurein a 15 L jacketed reactor with the jacket set at 50° C., internaltemperature 20° C. The additional batches were added to the reactor andconcentrated one at a time resulting in a slurry about 6 L in volume.The slurry was heated to 50° C., IPAc (6 L) was added and the mixturewas held at 60° C. for 1.5 h, cooled to 20° C. for 10 h, heated to 60°C. for 50 h, cooled to 20° C. in 5 h, then cooled to 5° C. and held for3 h. The mixture was filtered and the reactor and cake was washed with apremixed solution of IPAc (1 L) and MeTHF (1 L), precooled to 5° C. Thesolids were dried under nitrogen on the filter at 40° C. for 3 d toprovide the title compound (1059 g, 2.589 mol, 79% yield) as anoff-white solid. The material was further dried in a vacuum oven at50-60° C. for 8 h and at 27° C. for 2 d to provide the title compound(1043 g, 2.526 mol, 77% yield, 99% purity). HPLC Method A Retention time6.73 min.

Preparation 4: (4-(Benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate,potassium

(a)2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of 1-(benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (520 g, 1682mmol) and dioxane (5193 mL) was purged with nitrogen and thenbis(pinacolato)diboron (641 g, 2523 mmol) was added followed bypotassium acetate (495 g, 5046 mmol). The reaction mixture was purgedwith nitrogen; Pd(dppf)Cl₂ (41.2 g, 50.5 mmol) was added; the reactionmixture was purged with nitrogen, heated at 103° C. under nitrogen for 5h; and cooled to RT. The reaction mixture was concentrated by vacuumdistillation and partitioned between ethyl acetate (5204 mL) and water(5212 mL). The reaction mixture was filtered through Celite; the organiclayer was washed with brine (2606 mL) followed by solvent removal byvacuum distillation to provide crude product as a thick black oil (-800g).

The crude product was dissolved in DCM (1289 mL) and purified by silicagel chromatography (2627 g silica preslurried in hexane, eluted with 20%ethyl acetate in hexanes (10.35 L)). Solvent was removed by vacuumdistillation to yield a light yellow oil (600 g). HPLC Method BRetention time 33.74 min.

(b) (4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate, potassium

The product of the previous step (200 g, 561 mmol) was mixed withacetone (1011 mL) until complete dissolution and methanol (999 mL) wasadded followed by 3 M potassium hydrogen difluoride (307 g, 3930 mmol)dissolved in water (1310 mL).

The reaction mixture was stirred for 3.5 h. Most of the organic solventwas removed by vacuum distillation. Water (759 mL) was added and theresulting thick slurry was stirred for 30 min and filtered. The cake waswashed with water (506 mL) and the solids were dried on the filter for30 min. The solids were slurried in acetone (1237 mL) and stirred for 1h. The resulting slurry was filtered and the solids washed with acetone(247 mL).

The acetone solution was concentrated by vacuum distillation, and aconstant volume (2 L) was maintained by slow addition of toluene (2983mL) until all acetone and water had been distilled. The toluene solutionwas distilled to a thick yellow slurry by rotary evaporation, duringwhich time the products precipitated as white solids. An additionalportion of toluene (477 mL) was added to the mixture and stirred for 1h. The mixture was then filtered and rinsed with toluene (179 mL) anddried under vacuum at 50° C. for 24 h to provide the title compound (104g, 310 mmol, 55% yield) as a free-flowing, fluffy, slightly off-whitesolid. HPLC Method B Retention time 27.71 min.

Preparation 5:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

(a) 5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5 -fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine

A mixture of bis(pinacolato)diboron (250 g, 984 mmol) and IPA (1.88 L)was stirred to dissolution and then a solution of potassium hydrogendifluoride (538 g, 6.891 mol) in water (2.31 L) was added portion-wiseover 10 min. The reaction mixture was stirred for 1 h and filtered. Thegel-like solids were slurried with water (1.33 L) until the mixtureformed a clear hydrogel and then for another 45 min. The resultingsolids/gel were filtered, then reslurried in acetone (1.08 L), filtered,air dried on the filter for 30 min and dried overnight to provide afluffy white solid (196.7 g).

To a 5 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(135 g, 331 mmol),(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-trifluoroborate, potassium (133g, 397 mmol), and the white solid product of the previous step (40.5 g)followed by MeTHF (1.23 L) and MeOH (1.75 L). The resulting slurry wasdegassed three times with nitrogen. To the slurry was added a degassedsolution of cesium carbonate (431 g, 1.323 mol) in water (1.35 L). Theslurry was degassed twice, Pd (amphos)2C12 (11.71 g, 16.53 mmol) wasadded, the slurry was again degassed twice and the reaction mixture wasstirred at 67° C. overnight and cooled to 20° C. The layers wereseparated and back extracted with MeTHF (550 mL). The organic layerswere combined and concentrated by rotary evaporation until solidsprecipitated. MeTHF (700 mL) was added and the reaction mixture wasstirred at 65° C. The layers were separated and the aqueous phase backextracted with MeTHF (135 mL). The organic phases were combined andconcentrated to about 300 mL resulting in a thick orange slurry. To theslurry was added MeOH (270 mL) followed by 1M HCl (1.325 L) at 20° C.with rapid stirring. The reaction mixture was stirred for 5 min andwater (1 L) was added and the resulting slurry was stirred for 1 h. Thesolids were filtered, washed with water (150 mL), dried on the filterfor 10 min and at 45° C. under nitrogen for 16 h to provide the 2 HClsalt of the title compound (221.1 g, 351 mmol, 92.2% purity) as a lightyellow solid. HPLC Method B retention time 23.41 min.

Preparation 6:5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a 1 L flask was added5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine,2 HCl (40 g, 63.4 mmol) as a slurry in ethanol (348 mL) and 1.25 M HClin MeOH (101 mL) and water (17.14 mL). The reaction mixture was degassedwith nitrogen for 5 min and 10 wt % Pd/C, 50 wt % H₂O (4.05 g, 1.903mmol) was added. The reactor was sealed, purged with H₂ pressurized to1-2 psi. warmed to 50° C., and the reaction mixture was stirredovernight and filtered through Celite. The reactor and filter werewashed with methanol (100 mL).

The filtered solution was combined with the product of a second batch atthe 98 mmol scale and concentrated to 390 g. EtOAc (2.04 L) was addedslowly with stirring and then the solution was cooled to 5° C. withstirring. Solids were filtered, washed with EtOAc (510 mL), and driedovernight at 45° C. under nitrogen to provide the 2 HCl salt of thetitle compound (58 g, 80% yield) as an off-white solid. HPLC Method Bretention time 12.83 min.

Example 15-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHydrate

To a 125 mL flask was added NMP (19.23 mL) and5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,2 HCl (6 g, 13.32 mmol) with stirring followed by NMP (19.23 mL). Aceticacid (2.52 mL) was added and then 1-methylpiperidin-4-one (3.28 mL, 26.6mmol) was added in a single portion and the reaction mixture was stirredat 25° C. for 30 min and cooled to 15° C. Sodium triacetoxyborohydride(7.91 g, 37.3 mmol) was added and the external jacket was set to 20° C.after 20 min. After 3.5 h, total solution volume was 35 mL. The reactorwas washed with methanol (5 mL). Half the solution (17.5 mL) followed byhalf the methanol wash (2.5 mL) was added to a premixed solution ofmethanol (28 mL), ammonium hydroxide (17 mL, 270 mmol) and water (9 mL)maintaining the temperature below 5° C. Solids precipitated after 10min. The slurry was stirred for 30 min, ACN (60 mL) was added slowlyover 30 min and the slurry was stirred for 2 h at 0° C., filtered andrinsed with ACN. The solids were dried at 50° C. for 12 h to provide thetitle compound (2.95 g, 93.2% yield, (85.2% yield, corrected for watercontent)) as an off-white solid. HPLC Method C retention time 12.11 min

Example 2 Crystalline Hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(10 g, 21.07 mmol), prepared as in Example A, in DMSO (19.99 mL) wasadded ethanol (19.93 mL). Undissolved solids were removed by filtrationand half the DMSO solution was added to a stirred solution of 20% waterin methanol (30 mL). A slurry formed after 10 min, which was stirred atRT for 4 h and filtered. The resulting yellow solids were dried for 3 hat 50° C. under nitrogen. The solids were slurried in 20% water inacetone (110 mL) at 45° C. with stirring for 35 h, filtered, and washedwith 15% water in acetone and dried overnight to provide the titlecompound (4.40 g, 88% yield) as a light yellow solid.

Example 3 Crystalline Oxalate Hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

In a 20-mL glass vial, Compound 1 crystalline hydrate (248.5 mg) , theproduct of Example 2, and oxalic acid anhydrate (48.0 mg) were dissolvedin 1:1 tetrahydrofuran:water (5 mL). Acetonitrile (5 mL) was addedproducing a suspension. The resulting reaction mixture was stirred forone day at RT, filtered, washed with acetonitrile (2 mL), and driedunder ambient conditions overnight to provide the title compound.

Example 4 Crystalline Succinate Hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

In a 4-mL glass vial, Compound 1 crystalline hydrate (40 mg) andsuccinic acid (10 mg) were suspended in isopropanol (1 mL). The reactionmixture suspension was stirred for seven days at RT. The solids werefiltered, washed with isopropanol (0.5 mL), and dried under ambientconditions overnight to provide a crystalline succinate solvate. Theisolated succinate solvate solid was dried at 150° C. for 30 min undervacuum oven to provide a second solid form, which was equilibrated at80% to 90% relative humidity for one day at RT to provide the titlecompound.

Examples 5-7 Properties of the Solid Forms of the Invention

Samples of the crystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolof Example 3 and the crystalline hydrate of the succinate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolof Example 4 were analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Example 5 Powder X-Ray Diffraction

The powder X-ray diffraction patterns of FIG. 1 was obtained with aBruker D8-Advance X-ray diffractometer using Cu-Kα radiation (λ=1.54054Å) with output voltage of 45 kV and current of 40 mA. The instrument wasoperated in Bragg-Brentano geometry with incident, divergence, andscattering slits set to maximize the intensity at the sample. Formeasurement, a small amount of powder (5-25 mg) was gently pressed ontoa sample holder to form a smooth surface and subjected to X-rayexposure. The samples were scanned in 20-20 mode from 2° to 40° in 20with a step size of 0.02° and a scan speed of 0.30° seconds per step.The data acquisition was controlled by Bruker DiffracSuite measurementsoftware and analyzed by Jade software (version 7.5.1). The instrumentwas calibrated with a corundum standard, within±0.02° two-theta angle.Observed PXRD 2θ peak positions and d-spacings are shown in Tables 1 and2 for the crystalline oxalate hydrate and crystalline succinate hydrateof the invention, respectively.

TABLE 1 PXRD Data for the Crystalline Oxalate Hydrate 2θ d(Å) Area A %6.77 13.05 31716 41.0 11.56 7.65 6303.00 8.20 12.13 7.29 20994 27.213.54 6.53 77308 100.0 14.29 6.19 4903.00 6.30 16.96 5.22 9024 11.717.23 5.14 27774 35.9 17.72 5.00 19582 25.3 18.00 4.92 39472 51.1 18.554.78 31259 40.4 18.76 4.73 18293 23.7 19.51 4.55 14796 19.1 20.18 4.4011319 14.6 20.69 4.29 16629 21.5 21.38 4.15 14261 18.4 21.98 4.04 1862124.1 22.30 3.98 17504 22.6 23.63 3.76 14213 18.4 24.12 3.69 29375 38.024.34 3.65 19430 25.1 24.67 3.61 15460 20.0 27.05 3.29 20767 26.9 27.263.27 24154 31.2 28.85 3.09 8021 10.4 29.80 3.00 14992 19.4 30.13 2.9617939 23.2 31.05 2.88 7191 9.3

TABLE 2 PXRD Data for the Crystalline Succinate Hydrate 2θ d(Å) Area A %4.81 18.34 58400 25.80 9.66 9.14 92725 41.00 10.46 8.45 17225 7.60 13.456.58 5912 2.60 13.78 6.42 6010 2.70 14.93 5.93 93135 41.20 16.21 5.4624930 11.00 16.78 5.28 226066 100.00 17.45 5.08 49392 21.80 19.10 4.6453460 23.60 19.61 4.52 80964 35.80 21.20 4.19 70129 31.00 21.92 4.0551995 23.00 22.87 3.88 67007 29.60 24.77 3.59 81836 36.20 27.27 3.274553 2.00 28.09 3.17 18019 8.00 28.77 3.10 17372 7.70 30.68 2.91 52022.30 31.74 2.82 14150 6.30

Example 6 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10° C/minfrom 0° C. to 250° C. A representative DSC thermogram of the crystallineoxalate hydrate and crystalline succinate hydrate of the invention isshown in FIGS. 2 and 6, respectively.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300° C. The balance and furnacechambers were purged with nitrogen flow during use. A representative TGAtrace of the crystalline oxalate hydrate and crystalline succinatehydrate of the invention is shown in FIGS. 3 and 7, respectively.

Example 7 Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) measurement was performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, FL 33016).A weighed sample was used and the humidity was lowest possible value(close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (˜0%RH) for 120 minutes, followed bytwo cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C. A representative DMS trace for the crystallineoxalate hydrate and crystalline succinate hydrate of the invention isshown in FIGS. 4 and 8, respectively.

Biological Assays

5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(compound 1) has been characterized in the following biological assays.

Assay 1: Biochemical JAK Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

The serially diluted compound was pre-incubated with each of the fourJAK enzymes and the substrate in white 384-well microplates (Corning) atambient temperature for lh. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for lh before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pK₁ (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Compound 1 exhibited the following enzyme potency.

TABLE 2 JAK 1 JAK 2 JAK 3 Tyk2 pK_(i) pK_(i) pK_(i) pK_(i) 10.2 10.8 9.79.8

Assay 2: Cellular JAK Potency Assay: Inhibition of IL-13

The AlphaScreen JAM cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM

GlutaMAX (Life Technologies). On day 1 of the assay, cells were seededat a 7,500 cells/well density in white poly-D-lysine-coated 384-wellplates (Corning) with 25 μL medium, and were allowed to adhere overnightin the incubator. On day 2 of the assay, the medium was removed andreplaced with 12 μL of assay buffer (Hank's Balanced Salt Solution/HBSS,25 mM HEPES, and 1 mg/ml bovine serum albumin/BSA) containingdose-responses of test compounds. The compound was serially diluted inDMSO and then diluted another 1000-fold in media to bring the final DMSOconcentration to 0.1%. Cells were incubated with test compounds at 37°C. for 1 h, and followed by the addition of 12 μl of pre-warmed IL-13(80 ng/mL in assay buffer) for stimulation. After incubating at 37° C.for 30 min, the assay buffer (containing compound and IL-13) wasremoved, and 10 μL of cell lysis buffer (25 mM HEPES, 0.1% SDS, 1%NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mM EGTA, and supplement with CompleteUltra mini protease inhibitors and PhosSTOP from Roche Diagnostics). Theplates were shaken at ambient temperature for 30 min before the additionof detection reagents. A mixture of biotin-anti-pSTAT6 and anti-STAT6conjugated acceptor beads was added first and incubated at ambienttemperature for 2 h, followed by the addition of streptavidin conjugateddonor beads (Perkin Elmer). After a minimum of 2 h incubation, the assayplates were read on the EnVision plate reader. AlphaScreen luminescencesignals were recorded and utilized to calculate the percent inhibitionvalues based on DMSO and background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC50 values were determined from a4-parameter robust fit model with the Prism software. Results may alsobe expressed as the negative logarithm of the IC₅₀ value, pIC₅₀.Compound 1 exhibited a pIC₅₀ value of 8.2 in this assay.

Assay 3: Cellular JAK Potency Assay: Inhibition of IL-2/anti-CD3Stimulated IFNγ in human PBMCs

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated interferon gamma (IFNy) was measured in humanperipheral blood mononuclear cells (PBMCs) isolated from human wholeblood (Stanford Blood Center). Because IL-2 signals through JAK, thisassay provides a measure of JAK cellular potency.

(1) Human peripheral blood mononuclear cells (PBMC) were isolated fromhuman whole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10%

Heat Inactivated Fetal Bovine Serum (FBS, Life Technologies), 2 mMGlutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1×Pen/Strep (Life Technologies). Cells were seeded at 200,000 cells/wellin media (50 μL) and cultured for 1 h. Compounds were serially dilutedin DMSO and then diluted another 500-fold (to a 2x final assayconcentration) in media. Test compounds (100 μL/well) were added tocells, and incubated at 37° C., 5% CO₂ for 1 h, followed by the additionof IL-2 (R&D Systems; final concentration 100 ng/mL) and anti-CD3 (BDBiosciences; final concentration 1 μg/mL) in pre-warmed assay media (50μL) for 24 h.

(2) After cytokine stimulation, cells were centrifuged at 500 g for 5min and supernatants removed and frozen at −80° C. To determine theinhibitory potency of the test compound in response to IL-²/_(a)nti-CD3,supernatant IFNγ concentrations were measured via ELISA (R&D Systems).IC₅₀ values were determined from analysis of the inhibition curves ofconcentration of IFNy vs compound concentration. Data are expressed aspIC₅₀ (negative decadic logarithm IC₅₀) values. Compound 1 exhibited apIC₅₀ value of about 7.3 in this assay.

Assay 4: Cellular JAK Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in CD4+T cells

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated STATS phosphorylation was measured inCD4-positive (CD4+) T cells in human peripheral blood mononuclear cells(PBMCs) isolated from human whole blood (Stanford Blood Center) usingflow cytometry. Because IL-2 signals through JAK, this assay provides ameasure of JAK cellular potency.

CD4+T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT5 antibody (pY694, Clone 47, BD Biosciences)was used to detect STATS phosphorylation.

(1) The protocol of Assay 3 paragraph (1) was followed with theexception that the cytokine stimulation with anti-CD3 was performed for30 min instead of 24 h. (2) After cytokine stimulation, cells were fixedwith pre warmed fix solution (200 4; BD Biosciences) for 10 min at 37°C., 5% CO₂, washed twice with DPBS buffer (1 mL, Life Technologies), andresuspended in ice cold Perm Buffer III (1000 4, BD Biosciences) for 30min at 4° C. Cells were washed twice with 2% FBS in DPBS (FACS buffer),and then resuspended in FACS buffer (100 4) containing anti-CD4 PE (1:50dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 minat room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences). To determine the inhibitory potency of the testcompound in response to IL-²/_(a)nti-CD3, the median fluorescentintensity (MFI) of pSTAT5 was measured in CD4+ T cells. IC₅₀ values weredetermined from analysis of the inhibition curves of MFI vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC50) values. Compound 1 exhibited a pIC₅₀ value of about 7.7 in thisassay.

Assay 5: Cellular JAK Potency Assay: Inhibition of IL-4 StimulatedpSTAT6 in CD3+ T cells

The potency of the test compound for inhibition of interleukin-4 (IL-4)stimulated STAT6 phosphorylation was measured in CD3-positive (CD3+) Tcells in human peripheral blood mononuclear cells (PBMCs) isolated fromhuman whole blood (Stanford Blood Center) using flow cytometry. BecauseIL-4 signals through JAK, this assay provides a measure of JAK cellularpotency.

CD3+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD3 antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences)was used to detect STAT6 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors as in Assays 3 and 4. Cells were seeded at250,000 cells/well in media (200 4), cultured for 1 h and thenresuspended in assay media (50 μL) (RPMI supplemented with 0.1% bovineserum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep)containing various concentrations of test compounds. Compounds wereserially diluted in DMSO and then diluted another 500-fold (to a 2×final assay concentration) in assay media. Test compounds (50 μL) wereincubated with cells at 37° C., 5% CO₂ for 1 h, followed by the additionof IL-4 (50 μL) (R&D Systems; final concentration 20 ng/mL) inpre-warmed assay media for 30 min. After cytokine stimulation, cellswere fixed with pre-warmed fix solution (100 μL) (BD Biosciences) for 10min at 37° C., 5% CO₂, washed twice with FACS buffer (1 mL) (2% FBS inDPBS), and resuspended in ice cold Perm Buffer III (1000 μL) (BDBiosciences) for 30 min at 4° C. Cells were washed twice with FACSbuffer, and then resuspended in FACS buffer (100 μL) containing anti-CD3PE (1:50 dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5 dilution) for 60min at room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences).

To determine the inhibitory potency of the test compound in response toIL-4, the median fluorescent intensity (MFI) of pSTAT6 was measured inCD3+ T cells. IC₅₀ values were determined from analysis of theinhibition curves of MFI vs compound concentration. Data are expressedas pIC₅₀ (negative decadic logarithm IC₅₀). Compound 1 exhibited a pIC₅₀value of 8.1 in this assay.

Assay 6: Cellular JAK Potency Assay: Inhibition of IL-6 StimulatedpSTAT3 in CD3+ T cells

A protocol analogous to that of Assay 5 was used to determine thepotency of the test compound for inhibition of interleuken-6 (IL-6)stimulated STAT3 phosphorylation.

An Alexa Fluor 647 conjugated anti-pSTAT3 antibody (pY705, Clone 4/P, BDBiosciences) was used to detect STAT3 phosphorylation.

Compound 1 exhibited a pIC₅₀ value of 7.4 in this assay.

Assay 7: Cellular JAK Potency Assay: Inhibition of IFNγ-Induced pSTAT1

The potency of the test compound for inhibition of interferon gamma(IFNγ) stimulated STAT1 phosphorylation was measured in CD14-positive(CD14+) monocytes derived from human whole blood (Stanford Blood Center)using flow cytometry. Because IFNγ signals through JAK, this assayprovides a measure of JAK cellular potency.

Monocytes were identified using a fluorescein isothiocyanate (FITC)conjugated anti-CD14 antibody (Clone RM052, Beckman Coulter), and anAlexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, Clone 4a, BDBiosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Fetal Bovine Serum (FBS, LifeTechnologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (LifeTechnologies) and 1× Pen/Strep (Life Technologies). Cells were seeded at250,000 cells/well in media (200 4), cultured for 2 h and resuspended inassay media (50 4) (RPMI supplemented with 0.1% bovine serum albumin(Sigma), 2 mM Glutamax, 25 mM HEPES and 1X Penstrep) containing variousconcentrations of test compounds. The compound was serially diluted inDMSO and then diluted another 1000-fold in media to bring the final DMSOconcentration to 0.1%. Test compound dilutions were incubated with cellsat 37° C., 5% CO₂ for 1 h, followed by the addition of pre-warmed IFNγ(R&D Systems) in media (50 4) at a final concentration of 0.6 ng/mL for30 min. After cytokine stimulation, cells were fixed with pre-warmed fixsolution (100 μL) (BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with FACS buffer (1 mL) (1% BSA in PBS), resuspended in 1:10anti-CD14 FITC:FACS buffer (100 μL), and incubated at 4° C. for 15 min.Cells were washed once, and then resuspended in ice cold Perm Buffer III(BD Biosciences) (100 μL) for 30 min at 4° C. Cells were washed twicewith FACS buffer, and then resuspended in 1:10 anti-pSTAT1 Alexa Fluor647:FACS buffer (100 μL) for 30 min at RT in the dark, washed twice inFACS buffer, and analyzed using a LSRII flow cytometer (BD Biosciences).

To determine the inhibitory potency of the test compound, the medianfluorescent intensity (MFI) of pSTAT1 was measured in CD14+ monocytes.IC₅₀ values were determined from analysis of the inhibition curves ofMFI vs compound concentration. Data are expressed as pIC₅₀ (negativedecadic logarithm IC₅₀) values. Compound 1 exhibited a pIC₅₀ value ofabout 7.5 in this assay.

Assay 8: Pharmacokinetics in Plasma and Lung in Mouse

Plasma and lung levels of test compounds and ratios thereof weredetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Test compounds were individuallyformulated in 20% propylene glycol in pH 4 citrate buffer at aconcentration of 0.2 mg/mL and 50 uL of the dosing solution wasintroduced into the trachea of a mouse by oral aspiration. At varioustime points (typically 0.167, 2, 6, 24 hr) post dosing, blood sampleswere removed via cardiac puncture and intact lungs were excised from themice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungswere padded dry, weighed, and homogenized at a dilution of 1:3 insterile water. Plasma and lung levels of test compound were determinedby LC-MS analysis against analytical standards constructed into astandard curve in the test matrix. A lung to plasma ratio was determinedas the ratio of the lung AUC in μg hr/g to the plasma AUC in μg hr/mL,where AUC is conventionally defined as the area under the curve of testcompound concentration vs. time.

Compound 1 exhibited exposure in lung about 55 times greater thanexposure in plasma in mouse.

Assay 9: Murine (Mouse) model of IL-13 induced pSTAT6 induction in lungtissue

11-13 is an important cytokine underlying the pathophysiology of asthma(Kudlacz et al. Eur. J. Pharmacol, 2008, 582,154-161). IL-13 binds tocell surface receptors activating members of the Janus family of kinases(JAK) which then phosphorylate STAT6 and subsequently activates furthertranscription pathways. In the described model, a dose of IL-13 wasdelivered locally into the lungs of mice to induce the phosphorylationof STAT6 (pSTAT6) which is then measured as the endpoint.

Adult balb/c mice from Harlan were used in the assay. On the day ofstudy, animals were lightly anesthetized with isoflurane andadministered either vehicle or test compound (0.5 mg/mL, 50 μL totalvolume over several breaths) via oral aspiration. Animals were placed inlateral recumbency post dose and monitored for full recovery fromanesthesia before being returned to their home cage. Four hours later,animals were once again briefly anesthetized and challenged with eithervehicle or IL-13 (0.03 μg total dose delivered, 50 μL total volume) viaoral aspiration before being monitored for recovery from anesthesia andreturned to their home cage. One hour after vehicle or IL-13administration, lungs were collected for both pSTAT6 detection using ananti-pSTAT6 ELISA (rabbit mAb capture/coating antibody; mouse mAbdetection/report antibody: anti-pSTAT6-pY641; secondary antibody:anti-mouse IgG-HRP) and analyzed for total drug concentration asdescribed above in Assay 12.

Activity in the model is evidenced by a decrease in the level of pSTAT6present in the lungs of treated animals at 5 hours compared to thevehicle treated, IL-13 challenged control animals. The differencebetween the control animals which were vehicle- treated, IL-13challenged and the control animals which were vehicle-treated, vehiclechallenged dictated the 0% and 100% inhibitory effect, respectively, inany given experiment. Compound 1 exhibited about 60% inhibition of STAT6phosphorylation at 4 hours after IL-13 challenge.

Assay 10: Murine model of Alternaria alternata-induced eosinophilicinflammation of the lung

Airway eosinophilia is a hallmark of human asthma. Alternaria alternatais a fungal aeroallergen that can exacerbate asthma in humans andinduces eosinophilic inflammation in the lungs of mice (Havaux et al.Clin Exp Immunol. 2005, 139(2):179-88). In mice, it has beendemonstrated that alternaria indirectly activates tissue resident type 2innate lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and coordinateeosinophilic inflammation (Bartemes et al. J Immunol. 2012,188(3):1503-13).

Seven- to nine-week old male C57 mice from Taconic were used in thestudy. On the day of study, animals were lightly anesthetized withisoflurane and administered either vehicle or test compound (0.1-1.0mg/mL, 50 μL total volume over several breaths) via oropharyngealaspiration. Animals were placed in lateral recumbency post dose andmonitored for full recovery from anesthesia before being returned totheir home cage. One hour later, animals were once again brieflyanesthetized and challenged with either vehicle or alternaria extract(200 ug total extract delivered, 50 μL total volume) via oropharyngealaspiration before being monitored for recovery from anesthesia andreturned to their home cage. Forty-eight hours after alternariaadministration, bronchoalveolar lavage fluid (BALF) was collected andeosinophils were counted in the BALF using the Advia 120 HematologySystem (Siemens).

Activity in the model is evidenced by a decrease in the level ofeosinophils present in the BALF of treated animals at forty-eight hourscompared to the vehicle treated, alternaria challenged control animals.Data are expressed as percent inhibition of the vehicle treated,alternaria challenged BALF eosinophils response. To calculate percentinhibition, the number of BALF eosinophils for each condition isconverted to percent of the average vehicle treated, alternariachallenged BALF eosinophils and subtracted from one-hundred percent.Compound 1 exhibited about 88% inhibition of BALF eosinophil counts atforty-eight hours after alternaria challenge.

Assay 11: Murine model of LPS/G-CSF/IL-6/IFNγ Cocktail-Induced AirwayNeutrophilic Inflammation of the Lung Model

Airway neutrophilia is a hallmark of a range of respiratory disease inhumans. Compound 1 was tested in a model of neutrophilic airwayinflammation using a LPS/G-CSF/IL-6/IFNγ cocktail to induce airwayneutrophilia.

Seven- to nine-week old male Balb/C (wildtype) mice from JacksonLaboratory were used in the study. On the day of study, animals werelightly anesthetized with isoflurane and administered either vehicle ortest compound (1.0 mg/mL, 50 μL total volume over several breaths) viaoropharyngeal aspiration. Animals were placed in lateral recumbency postdose and monitored for full recovery from anesthesia before beingreturned to their home cage. One hour later, animals were once againbriefly anesthetized and challenged with either vehicle or LPS; 0.01mg/kg/G-CSF; 5 μg/IL-6; 5 μg/IFNγ; 5 μg (100 μL total volume) viaoropharyngeal aspiration (OA). Twenty-four hours after theLPS/G-CSF/IL-6/IFNg cocktail administration, bronchoalveolar lavagefluid (BALF) was collected and neutrophils were counted.

Upon OA treatment with compound 1, there was a statistically significantreduction of the airway neutrophils (84% compared to vehicle treatedmice), demonstrating that the blockade of JAK-dependent signaling haseffects on neutrophilic airway inflammation.

Assay 12: Ocular Pharmacokinetics in Rabbit Eyes

The objective of this assay was to determine the pharmacokinetics of thecompound 1 in rabbit ocular tissues.

Solution Formulation

The compound of the invention,5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(1) was dissolved in either 10% 2-hydroxypropyl-β-cyclodextrin to attaina target concentration of 4 mg/mL or in purified water to attain atarget concentration of 1 mg/mL . Bilateral intravitreal injection (50μL/eye) of the solution of test compound was administered to New Zealandwhite rabbits in two dose groups, 200 μg/eye and 50 μg/eye,respectively, for the cyclodextrin and water vehicle formulations,respectively. The test compound concentration was measured in oculartissues: vitreous, aqueous, retina/choroid and iris-ciliary body atpre-determined time points post injection (30 min, 4 h, 1 d, 3 d, 7 d,14 d). Two rabbits (four eyes) were dosed for each time point. In thevitreous tissue, compound 1 exhibited a two-phase decrease inconcentration characterized by an initial decrease in concentration witha half-life of approximately 12 hours and finally a terminal half-lifeof approximately 3.6 days. The compound was found to distribute quicklyinto the retinal and choroidal region as well and shows a similarpharmacokinetic profile as in the vitreous tissue.

Suspension Formulation

A suspension formulation was prepared by combining crystalline compound1 of Example 2 with 0.5% hydroxypropyl methylcellulose (HPMC E5)+0.02%Tween 80 to attain a target concentration of 10 mg/mL. Bilateralintravitreal injection (50 μL/eye) of the suspension of test compoundwas administered to New Zealand white rabbits (500 μg/eye). The testcompound concentration was measured in ocular tissues as in thesuspension formulation assay at 30 min, 2 wks, 4 wks, 6 wks, and 8 wkspost injection. The compound showed a linear decrease in drugconcentration in the vitreous from 30 min to 6 weeks with a clearancerate of approximately 3 μg/mL/day. The behavior is consistent with thesolubility of compound 1 in the vehicle and the ocular pharmacokineticbehavior in the solution formulation. The drug concentration in plasmawas measured and found to be at least 3 orders of magnitude lower thanthe concentration in vitreous tissue.

Assay 13: Pharmacodynamic Assay: Inhibition of IL6-induced pSTAT3 inRats

The ability of a single intravitreal administration of test compound toinhibit IL-6 induced pSTAT3 was measured in rat retina/choroidhomogenates.

Suspension formulations were prepared by combining crystalline compound1 of Example 2 with 0.5% hydroxypropyl methylcellulose (HPMC E5 LV),0.02% Tween 80, and 0.9% sodium chloride in purified water to attaintarget concentrations of 3 mg/mL and 10 mg/mL.

Female Lewis rats were intravitreally (IVT) dosed (5 μL per eye) withthe suspension formulations or with the drug vehicle. Three days later,IL-6 (Peprotech; 0.1 mg/mL; 5 μL per eye) or vehicle was intravitreallyadministered to induce pSTAT3.

Ocular tissues were dissected one hour after the second IVT injectionwith IL-6. The retina/choroid tissues were homogenized and pSTAT3 levelswere measured using an ELISA (Cell Signaling Technology). The percentinhibition of IL-6-induced pSTAT3 was calculated in comparison to thevehicle/vehicle and vehicle/IL-6 groups. Inhibition of greater than 100%reflects a reduction of pSTAT3 levels to below those observed in thevehicle/vehicle group.

With a 3 day pre-treatment prior to IL-6 challenge, the 15 μg dose andthe 50 μg dose of the compound of the invention administered by thesuspension formulation inhibited IL-6-induced pSTAT3 by 33% and 109%,respectively in the retina/choroid tissues.

Assay 14: Pharmacodynamic Assay: Inhibition of IFNγ-induced IP-10 inRabbits

The ability of a single intravitreal administration of test compound toinhibit interferon-gamma (IFNγ) induced IP-10 protein levels wasmeasured in rabbit vitreous and retina/choroid tissues.

Solution formulations at concentrations of 1 mg/mL and 4 mg/mL ofcompound 1 of Example 2 were prepared as in Assay 12. A suspensionformulation was prepared by combining crystalline compound 1 of Example2 with 0.5% hydroxypropyl methylcellulose (HPMC E5), 0.02% Tween 80, and9 mg/mL sodium chloride in purified water to attain a targetconcentration of 20 mg/mL.

Male, New Zealand White rabbits (Liveon Biolabs, India) were used forthe studies. Animals were acclimated after arrival at the researchfacilities (Jubilant Biosys Ltd., India). Each rabbit was given a totalof two intravitreal (IVT) injections with a total dose volume of 50 μLper eye. The first IVT injection (45 μL per eye) delivered test compoundor vehicle at a prescribed time point (i.e. 24 hours for the solutionformulations or 1 week for the suspension formulation). The second IVTinjection (5 μL per eye) delivered IFNγ (1 μg/eye; Stock solution 1mg/mL; Kingfisher Biotech) or vehicle for the induction of IP-10. Inbrief, on the day of the injections, rabbits were anesthetized with anintramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg).Once deeply anesthetized, each eye was rinsed with sterile saline andIVT injections were performed using a 0.5 mL insulin syringe (50units=0.5 mL) with a 31-gauge needle at the supra-nasal side of the botheyes by marking the position with a Braunstein fixed caliper (2 ¾″) 3.5mm from the rectus muscle and 4 mm from the limbus.

Tissues were collected 24 hours after the second IVT injection withIFNγ. Vitreous humor (VH) and retina/choroid tissues (R/C) werecollected and homogenized, and IP-10 protein levels were measured usinga rabbit CXCL10 (IP-10) ELISA kit (Kingfisher Biotech). The percentinhibition of IFNγ-induced IP-10 was calculated in comparison to thevehicle/vehicle and vehicle/IFNγ groups.

When dosed as a solution, with a 24 hour pre-treatment prior to the IFNγchallenge, 45 μg of compound 1 inhibited IFNγ-induced IP-10 by 70% and86% in the vitreous humor and retina/choroid tissue, respectively, while180 μg of the compound inhibited IFNγ-induced IP-10 by 91% and 100% inthe vitreous humor and retina/choroid tissue, respectively.

With a 1 week pre-treatment prior to the IFNγ challenge, the crystallinesuspension formulation of compound 1 inhibited IFNγ-induced IP-10 by100% in both the vitreous humor and retina/choroid tissues.

Assay 15: Inhibition of IFNy and IL-27 induced chemokines CXCL9 andCXCL10 in human 3D airway cultures

EpiAirway tissue cultures were obtained from Mattek (AIR-100). Cultureswere derived from asthmatic donors. In a cell culture insert, humanderived tracheal/bronchial epithelial cells were grown anddifferentiated on a porous membrane support, allowing an air-liquidinterface with warmed culture medium below the cells and a gaseous testatmosphere above. Tissues were cultured in maintenance media (Mattek,AIR-100-MM) in a 37° C., 5% CO2 humidified incubator. Four donors weretested. On Day 0, tissue cultures were treated with test compounds at 10μM, 1 μM and/or 0.1 μM. Compounds were diluted in dimethyl sulfoxide(DMSO, Sigma) to a final concentration of 0.1%. DMSO at 0.1% was used asa vehicle control. Test compounds were incubated with cultures for 1hour at 37° C., 5% CO₂, followed by the addition of pre-warmed mediacontaining IFNγ (R&D Systems) or IL-27 (R&D Systems) at a finalconcentration at 100 ng/ml. Tissue cultures were maintained for 8 days.Media was replaced every 2 days with fresh media containing compoundsand IFNγ or IL-27. On Day 8, tissue cultures and supernatants werecollected for analysis. Supernatant samples were assayed for CXCL10(IP-10) and CXCL9 (MIG) using luminex analysis (EMD Millipore). Data isexpressed as % Inhibition +/−standard deviation (±STDV). Percentinhibition was determined by compound inhibitory potency against IFNγ orIL-27 induced CXCL10 or CXCL9 secretion compared to vehicle treatedcells. Data is the average from 3 or 4 donors. Compound 1 was able toinhibit IFNγ induced CXCL10 secretion by 99%±2.0 (at 10 μM), 71%±19 (atμM) and 17%±12 (at 0.1 μM) when compared to vehicle control. Compound 1was able to inhibit IFNγ induced CXCL9 secretion by 100%±0.3 (at 10 μM),99%±0.9 (at 1 μM) and 74%±17 (at 0.1 μM) when compared to vehicle.Compound 1 was able to inhibit IL-27 induced CXCL10 secretion by 108%±11(at 10 μM), 98%±10 (at 1 μM) and 73%±8.5 (at 0.1 μM) when compared tovehicle control. Compound 1 was able to inhibit IL-27 induced CXCL9secretion by 100%±0 (at 10 μM), 95%±3.7 (at 1 μM) and 75%±3.5 (at 0.1μM) when compared to vehicle control.

Assay 16: IL-5 Mediated Eosinophil Survival Assay

The potency of the test compound for IL-5 mediated eosinophil survivalwas measured in human eosinophils isolated from human whole blood(AllCells). Because IL-5 signals through JAK, this assay provides ameasure of JAK cellular potency. Human eosinophils were isolated fromfresh human whole blood (AllCells) of healthy donors. Blood was mixedwith 4.5% Dextran (Sigma-Aldrich) in a 0.9% sodium chloride solution(Sigma-Aldrich). Red blood cells were left to sediment for 35 minutes.The leukocyte rich upper layer was removed and layered over Ficoll-Paque(GE Healthcare) and centrifuged at 600 g for 30 minutes. The plasma andmononuclear cell layers were removed before the granulocyte layer waslysed with water to remove any contaminating red blood cells.Eosinophils were further purified using a human eosinophil isolation kit(Miltenyi Biotec). A fraction of the purified eosinophils were incubatedwith anti-CD16 FITC (Miltenyi Biotec) for 10 minutes at 4° C. in thedark. Purity was analyzed using a LSRII flow cytometer (BD Biosciences).

Cells were cultured in a 37° C., 5% CO₂ humidified incubator in RPMI1640 (Life Technologies) supplemented with 10% Heat Inactivated FetalBovine Serum (FBS, Life Technologies), 2 mM Glutamax (LifeTechnologies), 25 mM HEPES (Life Technologies) and 1× Pen/Strep (LifeTechnologies). Cells were seeded at 10,000 cells/well in media (50 μL).The plate was centrifuged at 300 g for 5 minutes and supernatantsremoved. Compounds were serially diluted in DMSO and then dilutedanother 500-fold to a 2× final assay concentration in media. Testcompounds (50 μL/well) were added to cells, and incubated at 37° C., 5%CO₂ for 1 hour, followed by the addition of IL-5 (R&D Systems; finalconcentrations 1 ng/mL and 10 pg/ml) in pre-warmed assay media (50 μL)for 72 hours.

After cytokine stimulation, cells were centrifuged at 300 g for 5 minand washed twice with cold DPBS (Life Technologies). To access viabilityand apoptosis, cells were incubated with Propidium Iodide (Thermo FisherScientific) and APC Annexin V (BD Biosciences) and analyzed using aLSRII flow cytometer (BD Biosciences). IC₅₀ values were determined fromanalysis of the viability curves of percent cell viability vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. Compound 1 exhibited a pIC₅₀ value of 7.9±0.5 in thepresence of 10 pg/ml IL-5 and a pIC₅₀ value of 6.5±0.2 in the presenceof 1 ng/ml IL-5.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A crystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.2. The crystalline oxalate hydrate of claim 1, wherein the crystallineoxalate hydrate is characterized by a powder X-ray diffraction patterncomprising diffraction peaks at 20 values of 6.77±0.20, 12.13±0.20,13.54±0.20, 17.23±0.20, and 18.00±0.20.
 3. The crystalline oxalatehydrate of claim 2 wherein the powder X-ray diffraction pattern isfurther characterized by having two or more additional diffraction peaksat 20 values selected from 11.56±0.20, 14.29±0.20, 19.51±0.20,21.38±0.20, and 23.63±0.20.
 4. The crystalline oxalate hydrate of claim1, wherein the crystalline oxalate hydrate is characterized by a powderX-ray diffraction pattern in which the peak positions are substantiallyin accordance with the peak positions of the pattern shown in FIG.
 1. 5.The crystalline oxalate hydrate of claim 1 wherein the crystallineoxalate hydrate is characterized by a differential scanning calorimetrytrace recorded at a heating rate of 10° C. per minute which shows amaximum in endothermic heat flow at a temperature between 266° C. and276° C.
 6. The crystalline oxalate hydrate of claim 1, wherein thecrystalline oxalate hydrate is characterized by a differential scanningcalorimetry trace substantially in accordance with that shown in FIG. 2.7. A crystalline hydrate of the succinate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.8. The crystalline succinate hydrate of claim 7, wherein the crystallinesuccinate hydrate is characterized by a powder X-ray diffraction patterncomprising diffraction peaks at 20 values of 4.81±0.20, 9.66±0.20,14.93±0.20, and 16.78±0.20.
 9. The crystalline succinate hydrate ofclaim 8 wherein the powder X-ray diffraction pattern is furthercharacterized by having two or more additional diffraction peaks at 20values selected from 10.46±0.20, 16.21±0.20, 17.45±0.20, 22.87±0.20, and24.77±0.20.
 10. The crystalline succinate hydrate of claim 7, whereinthe crystalline succinate hydrate is characterized by a powder X-raydiffraction pattern in which the peak positions are substantially inaccordance with the peak positions of the pattern shown in FIG.
 5. 11.The crystalline succinate hydrate of claim 7 wherein the crystallinesuccinate hydrate is characterized by a differential scanningcalorimetry trace recorded at a heating rate of 10° C. per minute whichshows a maximum in endothermic heat flow at a temperature between 180°C. and 190° C.
 12. The crystalline succinate hydrate of claim 7, whereinthe crystalline succinate hydrate is characterized by a differentialscanning calorimetry trace substantially in accordance with that shownin FIG.
 6. 13. A pharmaceutical composition comprising the crystallineoxalate hydrate of claim 2 or the crystalline succinate hydrate of claim8, and a pharmaceutically-acceptable carrier.
 14. A method of preparinga crystalline hydrate of the oxalate salt of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,the method comprising: (a) dissolving a 1:1 mixture of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol:oxalic acid in a 1:1 mixture of tetrahydrofuran:water at roomtemperature, (b) adding a 1:1:2 mixture oftetrahydrofuran:water:acetonitrile to produce a suspension, (c) stirringthe suspension for about one day, and (d) isolating the crystallinehydrate of the oxalate salt from the suspension.
 15. A method oftreating a respiratory disease in a mammal, the method comprisingadministering to the mammal a pharmaceutical composition comprising thecrystalline oxalate hydrate of claim 2 or the crystalline succinatehydrate of claim 8, and a pharmaceutically-acceptable carrier.
 16. Themethod of claim 15 wherein the respiratory disease is asthma, chronicobstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathicpulmonary fibrosis, acute lung injury, acute respiratory distresssyndrome, bronchitis, emphysema or bronchiolitis obliterans.
 17. Themethod of claim 15 wherein the respiratory disease is bronchiolitisobliterans.
 18. The method of claim 15 wherein the respiratory diseaseis asthma or chronic obstructive pulmonary disease.
 19. The method ofclaim 18 wherein the respiratory disease is asthma.
 20. The method ofclaim 15 wherein the pharmaceutical composition is administered byinhalation.
 21. The method of claim 15 wherein the respiratory diseaseis a lung infection, a helminthic infection, pulmonary arterialhypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, orinfiltrative pulmonary disease.
 22. The method of claim 15 wherein therespiratory disease is drug-induced pneumonitis, fungal inducedpneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivitypneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathicacute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliteransorganizing pneumonia, or immune-checkpoint-inhibitor inducedpneumonitis.
 23. A method of treating an ocular disease in a mammal, themethod comprising administering to the eye of the mammal apharmaceutical composition comprising the crystalline oxalate hydrate ofclaim 2 or the crystalline succinate hydrate of claim 8, and apharmaceutically-acceptable carrier.